Fertility comparison of frozen bull semen stored in cryogenic deep freezer (-152 °C) and LN2 container.

The present study aimed to use cryogenic deep freezers that could be a feasible alternative for cryopreserved semen storage. A total of 284 straws from three Simmental bulls and 272 Simmental cows were used. The experimental group consisted of 151 semen straws that were stored at -152 °C for a week. Moreover, the control group consisted of 133 semen straws that were stored at -196 °C. Firstly, two samples per bull (n = 6) were examined in terms of sperm kinetic parameters by CASA. Furthermore, plasma membrane, acrosome integrity (PMAI) and high mitochondrial membrane potential (HMMP) were analyzed by flow cytometry. Then, artificial inseminations were performed on Simmental cows with 272 straws belonging to two groups. Then, 56th-day Non-return Rate (NRR56) was determined. All spermatological data were subjected to a linear mixed model. Chi-Square test was performed to NRR56 between storage temperature groups. Also, logistic regression analysis was used to examine the effect of bull, storage temperature and age of cows on pregnancy status. While age of cows was included in the final logistic regression model, effect of bull x storage temperature was not included because it was found as non-significant. The post-thaw PMOT and STR of cryopreserved bull semen, which was stored at -152 °C, had lower and statistically significant values (p < 0.05). However, frozen bull semen, which were stored at -152 °C, kept its fertility ability as which stored at -196 °C. Besides, NRR56 of semen stored at -152 °C and -196 °C were detected as 57.24% (83/145) and 55.91% (71/127), respectively (p > 0.05). Nevertheless, these results should be supplemented with more pre-freezing and post-thaw sperm quality analyses and more fertility data for increasing the accuracy of the method.

Quality assessment of frozen bull semen with the precursor A-kinase anchor protein 4 biomarker.

In this study, the quality of frozen bull semen was evaluated with the proAKAP4 level test. Sixty straws of frozen bull semen from various batches (n = 30) belonging to six bulls were used in the current study. The frozen bull semen samples were analysed in terms of proAKAP4 levels, sperm morphology and sperm movement parameters at hour 0 and hour 3 after thawing. The semen samples were divided into three groups according to the proAKAP4 levels: low concentration (<25 ng/10x106 spermatozoa), moderate concentration (25 to 39 ng/10x106 spermatozoa) and high concentration (≥40 ng/10x106 spermatozoa). A positive correlation was found between the proAKAP4 level and total motility (TM3 ), progressive motility (PM3 ), VSL3 and VCL3 values obtained after the third-hour thermoresistance test (p < .05). There was a negative correlation between the percentage of sperm abnormal tail and the proAKAP4 level (p < .01). In addition, it was observed that the semen samples with proAKAP4 concentrations of 25 ng/106 spermatozoa and higher preserved the TM3 and PM3 motility characteristics. In conclusion, the proAKAP4 has the potential to become a biomarker protein to evaluate in the quality analysis of frozen-thawed semen.

Effects of Spermine and Spermidine supplemented extenders on post-thaw Spermatological Parameters in Stallion Semen Cryopreservation.

In this study, the effects of polyamines, Spermine and Spermidine, on long-term preservation and post-thaw spermatological parameters were evaluated. Moreover, determination of the most suitable polyamine and its dose that can be added to standard extenders were aimed. Four adult Arabian stallions were used in the study. Five ejaculates were collected from each of four stallions via artificial vagina two days interval. Each ejaculate was divided into 13 aliquots. INRA96 (95,5%), egg yolk (2%), and glycerol (2,5%) were used as a control extender. Extenders of experimental groups were prepared with different doses of Spermine and Spermidine (0,1 mg/ml; 0,2 mg/ml; 0,4 mg/ml; 1 mg/ml; 2 mg/ml; 4 mg/ml). Stallion semen that were cryopreserved with Control and experimental extenders were evaluated in terms of Total Motility, Progressive Motility, Plasma Membrane Integrity, Capacitation Index, Acrosome Integrity and DNA Fragmentation Index. At the end of the evaluations, it was determined that 0,2 mg/ml Spermine and 0,4 mg/ml Spermidine showed better Total Motility and Progressive Motility, numerically. On the other hand, it was observed that 4 mg/ml Spermine and Spermidine had the lowest statistically significant values (p < 0,001). While statistically similar differences were obtained between groups in terms of the Plasma Membrane and Acrosome Integrity, it was determined that all experimental groups had lower and statistically significant values in terms of Capacitation and DNA Fragmentation Index (p < 0,001). As result, it was observed that the stallion semen cryopreservation success can be increased by the addition of 1-2 mg/ml Spermine that had effective protection on Capacitation and DNA Fragmentation Index without damaging other spermatological properties.

Companion animals get close to the toxic aspects of antropogenic world: cytotoxicity of phthalates and bisphenol A on dog testicular primary cells.

Phthalates, which are among the most abundant plasticizers, have detrimental effects on the reproductive system. Similar to human, dogs are prominently exposed to phthalates in daily routines at low concentrations; while toys, training devices and commercial dog foods are considered as the primary sources of exposure. This study aimed to reveal and compare the cytotoxic effects of selected phthalates (Benzyl butyl phthalate (BBP), Diethyl phthalate (DEP), Bis (2-ethylhexyl) phthalate (DEHP), Di-'isobutyl' phthalate (DIBP), Di-'isodecyl' phthalate (-DIDP) Di-'isononyl' phthalate (DINP), Dimethyl phthalate (DMP), Di-n-octyl phthalate (DNOP)), and Bisphenol A (BPA) following 24 h exposure on primary testicular parenchymal cells of dog in vitro at concentrations between 0.001 and 2.5 nM. According to cytotoxicity results, DEHP was found to be the most toxic phthalate with IC50 at 22.53 µM; while DMP was the least (169.17 nM). IC50 of BPA was 161.81 nM, less than the average (61.95 nM) of phthalates. In addition, dog primary testicular cells were found more susceptible to the high molecular weight phthalates (DNOP, DEHP, DINP, DIDP) than low molecular weight phthalates (DMP, DEP, DIBP, BBP). Further studies should focus on morphological, physiological and molecular differences to comprehend the mechanisms involved as well as decreasing the risk for impaired spermatogenesis caused by environmental toxicants in companion animal medicine.

The use of infrared thermography to detect the stages of estrus cycle and ovulation time in anatolian shepherd dogs.

BACKGROUND: The aim of the study is to evaluate the effectiveness of thermographic monitoring, using the temperature changes of perianal and perivulvar areas for the determination of estrus in Anatolian Shepherd bitches. Fifteen bitches were used in the study. Blood and vaginal smear samples were collected and thermographic monitoring of perianal and perivulvar areas were carried out starting from proestrus to early diestrus. Also, external signs of estrus were investigated. Smear samples were evaluated by light microscopy after Diff-Quik staining method and superficial and keratinized superficial cells were determined as percentage (S + KS%). Progesterone and luteinizing hormone measurements were done by radioimmunoassay. The difference in temperature between perianal and perivulvar areas was evaluated through thermographic images by FLIR ResearchIR Software. RESULTS: According to the results obtained from the study, differences between progesterone and S + KS% were statistically significant (P < 0,05). Although temperature showed increase and decrease with progesterone and S + KS%, the differences were not important statistically (P > 0,05). Serum luteinizing hormone levels did not sign any difference (P > 0,05). CONCLUSIONS: As a result, thermographic monitoring alone is not enough for estrus detection in Anatolian Shepherd bitches. However, it can be used to assist the actual estrus detection technique in terms of providing some foreknowledge by evaluating the differences in temperature.